EP1715341B1 - Method and apparatus for determining the concentrations of at least 2 ligands. - Google Patents

Method and apparatus for determining the concentrations of at least 2 ligands. Download PDF

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Publication number
EP1715341B1
EP1715341B1 EP05007284A EP05007284A EP1715341B1 EP 1715341 B1 EP1715341 B1 EP 1715341B1 EP 05007284 A EP05007284 A EP 05007284A EP 05007284 A EP05007284 A EP 05007284A EP 1715341 B1 EP1715341 B1 EP 1715341B1
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EP
European Patent Office
Prior art keywords
receptor
competitor
ligand
concentration
sample
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Not-in-force
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EP05007284A
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German (de)
French (fr)
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EP1715341A1 (en
Inventor
Holger Dr. Klapproth
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TDK Micronas GmbH
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TDK Micronas GmbH
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Priority to DE502005002994T priority Critical patent/DE502005002994D1/en
Priority to EP05007284A priority patent/EP1715341B1/en
Priority to US11/397,347 priority patent/US7598044B2/en
Publication of EP1715341A1 publication Critical patent/EP1715341A1/en
Application granted granted Critical
Publication of EP1715341B1 publication Critical patent/EP1715341B1/en
Priority to US12/558,750 priority patent/US20100003702A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/421—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions by moving the components in a convoluted or labyrinthine path
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the invention relates to a method according to the preamble of claim 1 and a device according to the preamble of claim 7.
  • Such a device is off US Pat. No. 6,197,503 B1 known. It has a flow-through measuring chamber which is bounded on its underside by an approximately plate-shaped substrate on which a plurality of test sites are arranged, on which receptors are immobilized. The receptors are each binding-specific for a particular ligand contained in a sample to be analyzed.
  • a semiconductor chip is provided, which has in each case an optical sensor at the individual test sites.
  • the device In order to excite the emission of luminescence radiation as a function of the binding of the ligands to the receptors, the device has an optical radiation source in whose emission area the receptors are arranged.
  • the sample is introduced through an inlet opening into the measuring chamber in such a way that the ligands contained in the sample can bind to the receptors.
  • a predetermined exposure time are not bound to a receptor components of the sample from the Measuring chamber removed.
  • the duration of action is chosen so that after removal of the unbound components, only part of the binding sites of a receptor present at the individual test sites is bound to the ligand which is specific for the receptor in question.
  • the ratio of the bound binding sites to the free binding sites depends on the concentration of the relevant ligand in the sample.
  • a solution is introduced into the measuring chamber containing detection antibodies which are marked with a marker.
  • the detection antibodies are binding-specific for the ligands and bind to them.
  • the solution is removed from the measuring chamber to irradiate the test sites with the aid of the radiation source with the excitation radiation.
  • the detection antibodies bound indirectly to the receptors via the ligands are excited by the excitation radiation to emit luminescence radiation whose wavelength differs from the wavelength of the excitation radiation.
  • a measuring signal which is dependent on the luminescence radiation and thus the concentration of the ligands in the sample is generated for each test site.
  • the device has the disadvantage that it allows a simultaneous determination of the individual concentration values of the different ligands contained in the sample only in a limited concentration range.
  • the ligands contained in the sample may show concentration differences of up to six decades. So that in each case a concentration value can be determined for such a sample both for the ligand with the lowest concentration and for the ligand with the highest concentration, without binding all available receptor binding sites to a ligand at one of the test sites and thus the corresponding measured value into the limit, at least two attempts are made.
  • a first test intended to determine the concentration of the highest concentration ligand will have a short exposure time and a second test to determine the concentration of the lowest concentration ligand will have a much longer exposure time chosen as the first attempt.
  • the disadvantage here is that a new semiconductor chip is needed for each experiment, which is why the measurement is still relatively expensive and labor-intensive. Unfavorable is also that a correspondingly large amount of sample is needed. When examining blood samples taken from a patient's finger with the aid of a puncturing tool, this can mean that blood must be removed from the finger in at least two places, which is particularly problematic for children.
  • a method for determining the concentrations of at least two ligands in a sample to be analyzed, wherein each ligand is capable of competing with a single competitor.
  • the competitor has two binding domains of which a first binding domain is capable of binding a first receptor and a second binding domain is capable of binding a second receptor.
  • the first receptor is first immobilized on a solid phase substrate at a first test site and the second receptor is immobilized at a second test site.
  • the sample, the competitor and the solid phase consisting of the substrate and the first and second receptors are brought together such that the first ligand and / or the competitor bind to the first receptor and the second ligand and / or the competitor to the second receptor can.
  • components of the mixture that are not bound to an immobilized receptor are removed from the substrate.
  • a first measurement signal dependent on the quantity of the competitor bound to the first receptor and a second measurement signal dependent on the amount of the competitor bound to the second receptor are generated.
  • the first measurement signal is related to the amount of the first ligand and the second measurement signal to the amount of the second ligand in the sample.
  • This object is achieved with respect to the method in that a compound specific for the second receptor competitor is so mixed with the sample that the competitor is present in a predetermined concentration in the mixture thus obtained, that the mixture is brought into contact with the substrate, that the first ligand can bind to the first receptor and the second ligand and / or the competitor to the second receptor, then that components of the mixture which are not bound to an immobilized receptor are removed from the substrate, then one of the Concentration of the first ligand bound to the first receptor dependent first measurement signal and dependent on the concentration of the bound to the second receptor competitor second measurement signal are generated, and wherein with the aid of the first measurement signal, the concentration of the first ligand in the sample and with the aid of the second Measuring signal and the known Konzentrati on the competitor in the mixture, the concentration of the second ligand in the sample can be determined.
  • the ligands and / or receptors may be nucleic acids or derivatives thereof (DNA, RNA PNA, LNA, oligonucleotides, plasmids, chromosomes), peptides, proteins (enzyme, protein, oligopeptides, cellular receptor proteins and their complexes, peptide hormones, antibodies and their fragments), Carbohydrates and their derivatives, in particular glycosylated proteins and glycosides, fats, fatty acids and / or lipids.
  • the competitive assay by combining the competitive assay with a non-competitive assay in the same measurement chamber, it is possible to determine the concentrations of individual ligands in a single experiment for samples containing multiple ligands that differ greatly in their concentration.
  • concentration of the competitor in the mixture consisting of the sample and the competitor and the duration of exposure during which the mixture is in contact with the receptors are adjusted to the concentration ranges to be checked for the individual ligands so that after removal of the not a receptor bound components of the mixture at all test sites only a part of the binding sites binds receptors to a ligand, if this is contained in the concentration range to be examined in the sample.
  • the concentration of the competitor in the mixture is preferably selected to correspond to a limit of the concentration of the second ligand from which a statement such as e.g. good / bad "or” sick / healthy. "With the method according to the invention ligand concentrations in a range of g / l to ug / l can be detected side by side in a flow cell or similar measuring chamber.
  • a detection antibody labeled with a first marker for the first ligand is brought into contact with the first test site, after which markers not bound to an immobilized receptor are removed from the substrate, and then the first measurement signal in Dependent on the concentration of the first marker is generated.
  • the concentration of the first ligand can therefore be measured with the aid of a sandwich Elisa.
  • the competitor is labeled with a second marker, wherein after removing the components of the mixture not bound to an immobilized receptor from the substrate, the second measurement signal is generated as a function of the concentration of the second marker bound to the second receptor ,
  • the second marker may coincide with the first marker.
  • the first marker and / or second marker is (are) an enzyme if the enzyme is at least two during the detection of the measurement signals Chemicals is brought into contact, between which in the presence of the enzyme, a chemical redox reaction occurs, and when the first measurement signal and / or the second measurement signal is generated by measuring a redox potential (be).
  • the redox potential can be measured with the aid of an ISFET.
  • the first marker and / or second marker is irradiated during the detection of the measurement signals with an excitation radiation which excites the marker (s) for emitting a luminescence radiation, wherein the first measurement signal and / or the second Measuring signal is generated by measuring the luminescence of the marker in question (be).
  • the excitation radiation can be generated with the aid of a light source, for example a light-emitting diode and / or a xenon lamp.
  • the first and second markers preferably the same excitation radiation is used.
  • the first and second markers contain different dyes, such as e.g. Cy3 and Cy5, and that these dyes are excited at different wavelengths.
  • the first test site and / or second test cell is brought into contact with a chemiluminescent substrate during the detection of the measurement signals, depending on the binding of the first ligand to the first receptor and / or depending on the binding is excited by the competitor to the second receptor for emitting a luminescence, wherein the first measurement signal and / or the second measurement signal is generated by measuring the luminescence at the relevant test site (are).
  • the luminescence radiation is thereby generated without excitation radiation by chemical means.
  • the above object is achieved with respect to the device in that it comprises a mixing device, which is connected for mixing the sample with a binding agent for the second receptor specific competitor with a feed opening for the sample and a competitor-containing receiving space, that the mixing device is a discharge opening for the mixture formed from the sample and the competitor, which is connected to the inlet opening of the measuring chamber, that the mixing device is designed such that the competitor is present in a predetermined concentration in the mixture, that the second sensor is designed to detect a second measurement signal dependent on the concentration of the competitor bound to the second receptor and connected to an evaluation device for determining the concentration of the second ligand in the sample from the second measurement signal and the concentration of the competitor in the mixture is trained.
  • the sample is thus mixed before entering the measuring chamber by means of the mixing device with a binding agent specific for the second receptor competitor, so that in the mixing chamber for the second ligand a competitive assay and simultaneously for the first ligand a non-competitive assay can be performed.
  • a binding agent specific for the second receptor competitor a binding agent specific for the second receptor competitor
  • the competitor is stabilized in a gel-like, doughy or solid form in the receiving space, preferably such that it adheres to a boundary wall of the receiving space, and if the receiving space for releasing the competitor in the sample is designed as a flow mixing chamber, via which the Supply port for the sample is connected to the inlet opening of the measuring chamber.
  • the device is therefore particularly easy to handle.
  • the stabilization of the competitor preferably comprises at least one non-reducing disaccharide and at least one protein or polypeptide of the LEA class.
  • the non-reducing disaccharide may be selected from the group consisting of trehalose (D-glucopyranosyl-D-glucopyranose), sucrose ( ⁇ -D-fructofuranosyl- ⁇ -D-glucopyranoside) and derivatives thereof.
  • trehalose D-glucopyranosyl-D-glucopyranose
  • sucrose ⁇ -D-fructofuranosyl- ⁇ -D-glucopyranoside
  • derivatives thereof such stabilization is in WO 2004/004455 A2 described.
  • the present in stabilized form competitor is stable for a long time.
  • the flow mixing chamber has a mixer structure which is designed such that the mixture is deflected in alternately opposite directions as it flows through the fürftussmischhunt, and that the mixer structure between the present in gel, doughy or solid form Competitor and the inlet opening of the measuring chamber is arranged.
  • the mixer can be a so-called Möbius mixer, which can be produced with methods of microsystems technology with very compact dimensions.
  • the sensors are optical sensors, wherein the first receptor for detecting a first luminescence radiation generated as a function of the binding of the first ligand to the first receptor preferably directly on the first sensor and / or the second receptor for detecting a in Depending on the binding of the competitor to the second receptor generated second luminescent radiation is preferably arranged directly on the second sensor (are).
  • the luminescence radiation generated at the receptors can then be coupled directly into the sensor (s) without the detour via a converging lens.
  • the competitor is marked with a marker which can be excited by irradiation with an excitation radiation for emitting the luminescence radiation
  • the device for irradiating the second test site with the excitation radiation has at least one radiation source, and if the second sensor is sensitive to the luminescence radiation and is insensitive to the excitation radiation.
  • the radiation source can also be arranged outside the measuring chamber, if it has at least one wall region which is permeable to the excitation radiation.
  • a wall of the measuring chamber 2 is formed by a semiconductor substrate on which a first receptor 6 is immobilized on a first test site 5 and a second receptor 8 is immobilized on a second test site 7 spaced laterally therefrom.
  • the first receptor 6 is for a first, in the analyzed Sample contained ligand 9 and the second receptor 8 for a second, contained in the sample ligand binding specific.
  • the second ligand has a greater concentration in the sample than the first ligand 9.
  • a first optical sensor 10 is integrated directly below the first receptor 6 and a second optical sensor 11 is integrated into the semiconductor substrate at the second test site 7 directly below the second receptor 8.
  • a third optical sensor 12 is arranged in the wall of the measuring chamber, which is not covered with a receptor and serves as a reference value transmitter.
  • the sensors 10, 11, 12 may be photodiodes, for example.
  • the device 1 further comprises a mixing device 1 3, which connects the inlet opening 3 of the measuring chamber 2 with a feed opening 14.
  • the mixing means 13 serves to mix the sample with a second receptor-specific competitor 15 which is bound to an enzyme 16, such as an enzyme.
  • HRP horseradish peroxidase
  • the sample is introduced, for example with the aid of a pipette or a pump through the feed opening 14 into a receiving space 16 of the mixing device 13.
  • the marked competitor 15 is arranged in a stabilized form relative to the feed opening 14 such that the sample comes into contact with the competitor 15 or the associated enzyme 16 during and / or after being introduced into the receiving space 17 mixed with the competitor-enzyme complexes.
  • the mixing device 1 3 has a mixer structure 18 configured as a Möbius mixer, which with its one end having the receiving space 17 and with their other, a discharge opening for the mixture formed from the sample and the competitor-enzyme complexes End is connected via a channel 19 with the inlet opening 3 of the measuring chamber 2.
  • the mixing device 13 is configured in such a way that the competitor 15 is present in the mixing chamber 2 at a predetermined concentration in the mixture corresponding to a limit value to be measured. This is achieved in that the measuring chamber 2 and the mixing device 13 have a predetermined volume, and that the amount of competitor 15 stabilized in the receiving space 17 is selected such that the desired concentration is established, when the competitor 15 is mixed with the sample to a mixture having the predetermined volume.
  • a predetermined first period of time is waited to give the ligands contained in the mixture and the competitor 15 the possibility to bind to each of them To bind receptors 6, 8.
  • the first time period is selected such that in each case only part of the free binding sites of the receptors 6, 8 present at the individual test sites 5, 7 bind to a ligand.
  • Fig. 2 is the binding of a molecule of the first ligand 9 to the first receptor 6 and in FIG Fig. 3 the binding of a competitor-enzyme complex to the second receptor 8 is shown schematically.
  • a rinsing liquid is passed through the measuring chamber 2 via the feed opening 14, the inlet opening 3 and the outlet opening 4, which removes the components of the mixture formed from the measuring chamber 2 which are not bound to a receptor 6, 8.
  • a solution is introduced into the measuring chamber 2 which contains a detection antibody 20 which is specific for the first ligand 9 and which is bound with an enzyme 21, e.g. HRP, marked.
  • an enzyme 21, e.g. HRP e.g. HRP
  • a predetermined second time period is waited, which is selected so that almost all bound to the first receptor 6 molecules of the first ligand 9 each bind to a molecule of the detection antibody 20 and thereby indirectly with the Enzyme 21 can be marked.
  • the rinsing liquid is again passed through the measuring chamber 2, in order not to remove detection antibodies 20 bound to a first ligand from the measuring chamber 2.
  • a chemiluminescent substrate is introduced into the measuring chamber 2 containing hydrogen peroxide and a chemiluminophore such as luminol.
  • a chemiluminophore such as luminol.
  • the enzymes 16, 21 with the Hydrogen peroxide come into contact, free oxygen radicals are cleaved from the hydrogen peroxide, whereby the chemiluminophore is chemically decomposed under the emission of luminescence radiation.
  • the luminescence radiation is thus generated on the one hand as a function of the binding of the first ligand to the first receptor 6 and on the other hand as a function of the binding of the competitor 15 to the second receptor 8 in the measuring chamber 2.
  • the optical sensors 10, 11 are sensitive to the luminescence radiation and arranged relative to the receptors 6, 8 such that they respectively detect only the luminescence radiation generated at their associated test site 5, 7, but not the luminescence radiation at the respective other test site 7, 5 is generated.
  • the evaluation device can be integrated as an electrical circuit in the substrate or the wall of the measuring chamber 2.
  • Fig. 4 and 5 For example, readings taken with a direct ELISA and a sandwich ELISA are plotted as a function of time.
  • a luminescence radiation is generated in the case of the ELISA.
  • the ligands are for this purpose marked with the aid of a marker, such as the dye Cy5, and irradiated with an excitation radiation, in which the marker is excited to emit luminescence radiation. It can be clearly seen that the increase in the measured values becomes flatter with decreasing concentration of the ligands in the sample.

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Description

Die Erfindung betrifft ein Verfahren nach dem Oberbegriff von Anspruch 1 und eine Vorrichtung nach dem Oberbegriff von Anspruch 7.The invention relates to a method according to the preamble of claim 1 and a device according to the preamble of claim 7.

Eine derartige Vorrichtung ist aus US 6 197 503 B1 bekannt. Sie weist eine Durchfluss-Messkammer auf, die an ihrer Unterseite durch ein etwa plattenförmiges Substrat begrenzt ist, auf dem mehrere Teststellen angeordnet sind, an denen Rezeptoren immobilisiert sind. Die Rezeptoren sind jeweils für einen bestimmten, in einer zu analysierenden Probe enthaltenden Liganden bindungsspezifisch. An der Rückseite des Substrats ist ein Halbleiterchip vorgesehen, der an den einzelnen Teststellen jeweils einen optischen Sensor aufweist. Zur Anregung der Emission von Lumineszenzstrahlung in Abhängigkeit von der Bindung der Liganden an die Rezeptoren weist die Vorrichtung eine optische Strahlungsquelle auf, in deren Abstrahlbereich die Rezeptoren angeordnet sind. Zum Messen der Konzentration der Liganden wird die Probe durch eine Einlassöffnung hindurch derart in die Messkammer eingebracht, dass die in der Probe enthaltenen Liganden an die Rezeptoren binden können. Nach Ablauf einer vorbestimmten Einwirkungsdauer werden nicht an einen Rezeptor gebundenen Bestandteile der Probe aus der Messkammer entfernt. Die Einwirkungsdauer wird so gewählt, dass nach dem Entfernen der ungebundnen Bestandteile nur ein Teil der an den einzelnen Teststellen jeweils vorhandenen Bindungsstellen eines Rezeptors an den für den betreffenden Rezeptor bindungsspezifischen Liganden gebunden ist. Dabei ist das Verhältnis der gebundenen Bindungsstellen zu den freien Bindungsstellen von der Konzentration des betreffenden Liganden in der Probe abhängig.Such a device is off US Pat. No. 6,197,503 B1 known. It has a flow-through measuring chamber which is bounded on its underside by an approximately plate-shaped substrate on which a plurality of test sites are arranged, on which receptors are immobilized. The receptors are each binding-specific for a particular ligand contained in a sample to be analyzed. At the back of the substrate, a semiconductor chip is provided, which has in each case an optical sensor at the individual test sites. In order to excite the emission of luminescence radiation as a function of the binding of the ligands to the receptors, the device has an optical radiation source in whose emission area the receptors are arranged. For measuring the concentration of the ligands, the sample is introduced through an inlet opening into the measuring chamber in such a way that the ligands contained in the sample can bind to the receptors. After a predetermined exposure time are not bound to a receptor components of the sample from the Measuring chamber removed. The duration of action is chosen so that after removal of the unbound components, only part of the binding sites of a receptor present at the individual test sites is bound to the ligand which is specific for the receptor in question. The ratio of the bound binding sites to the free binding sites depends on the concentration of the relevant ligand in the sample.

In einem weiteren Verfahrensschritt wird eine Lösung in die Messkammer eingebracht, die Nachweisantikörper enthält, die mit einem Marker markiert sind. Die Nachweisantikörper sind für die Liganden bindungsspezifisch und binden an diese. Danach wird die Lösung aus der Messkammer entfernt, um die Teststellen mit Hilfe der Strahlungsquelle mit der Anregungsstrahlung zu bestrahlen. Die über die Liganden indirekt an die Rezeptoren gebundenen Nachweisantikörper werden durch die Anregungsstrahlung zur Aussendung einer Lumineszenzstrahlung angeregt, deren Wellenlänge sich von der Wellenlänge der Anregungsstrahlung unterscheidet. Mit Hilfe der optischen Sensoren, die für die Lumineszenzstrahlung empfindlich und für die Anregungsstrahlung unempfindlich sind, wird für jede Teststelle jeweils ein von der Lumineszenzstrahlung und somit der Konzentration der Liganden in der Probe abhängiges Messsignal erzeugt.In a further method step, a solution is introduced into the measuring chamber containing detection antibodies which are marked with a marker. The detection antibodies are binding-specific for the ligands and bind to them. Thereafter, the solution is removed from the measuring chamber to irradiate the test sites with the aid of the radiation source with the excitation radiation. The detection antibodies bound indirectly to the receptors via the ligands are excited by the excitation radiation to emit luminescence radiation whose wavelength differs from the wavelength of the excitation radiation. With the aid of the optical sensors, which are sensitive to the luminescence radiation and insensitive to the excitation radiation, a measuring signal which is dependent on the luminescence radiation and thus the concentration of the ligands in the sample is generated for each test site.

Die Vorrichtung hat den Nachteil, dass sie nur in einem begrenzten Konzentrationsbereich eine gleichzeitige Bestimmung der einzelnen Konzentrationswerte der unterschiedlichen, in der Probe enthaltenen Liganden ermöglicht. Bei bestimmten Proben, wie zum Beispiel Blutproben, kann es jedoch vorkommen, dass die in der Probe enthaltenen Liganden Konzentrationsunterschiede von bis zu sechs Dekaden aufweisen. Damit bei einer solchen Probe sowohl für den Liganden mit der geringsten Konzentration als auch für den Liganden mit der höchsten Konzentration jeweils ein Konzentrationswert bestimmt werden kann, ohne dass es an einer der Teststellen alle vorhandenen Rezeptor-Bindungsstellen an einen Liganden binden und somit der entsprechende Messwert in die Begrenzung gerät, werden mindestens zwei Versuche durchgeführt. Bei einem ersten, zur Bestimmung der Konzentration des den höchsten Konzentrationswert aufweisenden Liganden vorgesehenen Versuch wird eine geringe Einwirkungsdauer und bei einem zweiten, zur Bestimmung der Konzentration des den niedrigsten Konzentrationswert aufweisenden Liganden vorgesehenen Versuch, wird eine wesentlich größere Einwirkungsdauer gewählt als bei dem ersten Versuch. Nachteilig ist dabei, dass für jeden Versuch jeweils ein neuer Halbleiterchip benötigt wird, weshalb die Messung noch relativ teuer und arbeitsaufwändig ist. Ungünstig ist außerdem, dass eine entsprechend große Probenmenge benötigt wird. Bei der Untersuchung von Blutproben, die mit Hilfe eines Stechwerkzeugs aus dem Finger eines Patienten entnommen wird, kann dies bedeuten, dass an mindestens zwei Stellen Blut aus dem Finger entnommen werden muss, was besonders bei Kindern problematisch ist.The device has the disadvantage that it allows a simultaneous determination of the individual concentration values of the different ligands contained in the sample only in a limited concentration range. However, for certain samples, such as blood samples, it is possible that the ligands contained in the sample may show concentration differences of up to six decades. So that in each case a concentration value can be determined for such a sample both for the ligand with the lowest concentration and for the ligand with the highest concentration, without binding all available receptor binding sites to a ligand at one of the test sites and thus the corresponding measured value into the limit, at least two attempts are made. A first test intended to determine the concentration of the highest concentration ligand will have a short exposure time and a second test to determine the concentration of the lowest concentration ligand will have a much longer exposure time chosen as the first attempt. The disadvantage here is that a new semiconductor chip is needed for each experiment, which is why the measurement is still relatively expensive and labor-intensive. Unfavorable is also that a correspondingly large amount of sample is needed. When examining blood samples taken from a patient's finger with the aid of a puncturing tool, this can mean that blood must be removed from the finger in at least two places, which is particularly problematic for children.

Aus WO 92/18866 A1 ist ferner ein Verfahren zum Bestimmen der Konzentrationen von mindestens zwei Liganden in einer zu analysierenden Probe bekannt, bei dem jeder Ligand zum Konkurrieren mit einem einzigen Kompetitor fähig ist. Der Kompetitor hat zwei Bindungsdomönen, von denen eine erste Bindungsdomäne zum Binden eines ersten Rezeptors und eine zweite Bindungsdomäne zum Binden eines zweiten Rezeptors fähig sind. Bei dem Verfahren werden zunächst auf einem Festphasen-Substrat an einer ersten Teststelle der erste Rezeptor und an einer zweiten Teststelle der zweite Rezeptor immobilisiert. Dann werden die Probe, der Kompetitor und die aus dem Substrat sowie den ersten und zweiten Rezeptoren bestehende Festphase derart zusammengebracht, dass der erste Ligand und/oder der Kompetitor an den ersten Rezeptor und der zweite Ligand und/oder der Kompetitor an den zweiten Rezeptor binden können. Danach werden Bestandteile des Gemischs, die nicht an einen immobilisierten Rezeptor gebunden sind, von dem Substrat entfernt. Dann werden ein von der Menge des an den ersten Rezeptor gebundenen Kompetitors abhängiges erstes Messsignal und ein von der Menge des an den zweiten Rezeptor gebundenen Kompetitors abhängiges zweites Messsignal erzeugt. Das erste Messsignal wird mit der Menge des ersten Liganden und das zweite Messsignal mit der Menge des zweiten Liganden in der Probe in Beziehung gesetzt. Auch dieses Verfahren hat den Nachteil, dass es nur in einem begrenzten Konzentrationsbereich eine gleichzeitige Bestimmung der einzelnen Konzentrationswerte der unterschiedlichen, in der Probe enthaltenen Liganden ermöglicht.Out WO 92/18866 A1 Further, a method is known for determining the concentrations of at least two ligands in a sample to be analyzed, wherein each ligand is capable of competing with a single competitor. The competitor has two binding domains of which a first binding domain is capable of binding a first receptor and a second binding domain is capable of binding a second receptor. In the method, the first receptor is first immobilized on a solid phase substrate at a first test site and the second receptor is immobilized at a second test site. Then the sample, the competitor and the solid phase consisting of the substrate and the first and second receptors are brought together such that the first ligand and / or the competitor bind to the first receptor and the second ligand and / or the competitor to the second receptor can. Thereafter, components of the mixture that are not bound to an immobilized receptor are removed from the substrate. Then, a first measurement signal dependent on the quantity of the competitor bound to the first receptor and a second measurement signal dependent on the amount of the competitor bound to the second receptor are generated. The first measurement signal is related to the amount of the first ligand and the second measurement signal to the amount of the second ligand in the sample. This method also has the disadvantage that only in a limited concentration range it is possible to determine simultaneously the individual concentration values of the different ligands contained in the sample.

Es besteht deshalb die Aufgabe, eine Vorrichtung und ein Verfahren der eingangs genannten Art zu schaffen, die eine einfache, schnelle und kostengünstige Messung der Konzentration mehrerer Liganden ermöglichen, von denen vermutet wird, dass sie in einer zu untersuchenden Probe enthalten sind.It is therefore an object to provide a device and a method of the type mentioned, which allow a simple, fast and inexpensive measurement of the concentration of multiple ligands, which is suspected to be contained in a sample to be examined.

Diese Aufgabe wird bezüglich des Verfahrens dadurch gelöst, dass ein für den zweiten Rezeptor bindungsspezifischer Kompetitor derart mit der Probe vermischt wird, dass der Kompetitor in einer vorgegebenen Konzentration in dem so erhaltenen Gemisch vorliegt, dass das Gemisch derart mit dem Substrat in Kontakt gebracht wird, dass der erste Ligand an den ersten Rezeptor und der zweite Ligand und/oder der Kompetitor an den zweiten Rezeptor binden können, dass danach Bestandteile des Gemischs, die nicht an einen immobilisierten Rezeptor gebunden sind, von dem Substrat entfernt werden, dass dann ein von der Konzentration der an den ersten Rezeptor gebundenen ersten Liganden abhängiges erstes Messsignal und ein von der Konzentration des an den zweiten Rezeptor gebundenen Kompetitors abhängiges zweites Messsignal erzeugt werden, und wobei mit Hilfe des ersten Messsignals die Konzentration des ersten Liganden in der Probe und mit Hilfe des zweiten Messsignals und der bekannten Konzentration des Kompetitors in dem Gemisch die Konzentration des zweiten Liganden in der Probe bestimmt werden. Die Liganden und/oder Rezeptoren können Nukleinsäuren oder Derivate davon (DNA, RNA PNA, LNA, Oligonukleotide, Plasmide, Chromosomen), Peptide, Proteine (Enzym, Protein, Oligopeptide, zelluläre Rezeptorproteine und deren Komplexe, Peptidhormone, Antikörper und deren Fragmente), Kohlenhydrate und deren Derivate, insbesondere glykosylierte Proteine und Glycoside, Fette, Fettsäuren und/oder Lipide umfassen.This object is achieved with respect to the method in that a compound specific for the second receptor competitor is so mixed with the sample that the competitor is present in a predetermined concentration in the mixture thus obtained, that the mixture is brought into contact with the substrate, that the first ligand can bind to the first receptor and the second ligand and / or the competitor to the second receptor, then that components of the mixture which are not bound to an immobilized receptor are removed from the substrate, then one of the Concentration of the first ligand bound to the first receptor dependent first measurement signal and dependent on the concentration of the bound to the second receptor competitor second measurement signal are generated, and wherein with the aid of the first measurement signal, the concentration of the first ligand in the sample and with the aid of the second Measuring signal and the known Konzentrati on the competitor in the mixture, the concentration of the second ligand in the sample can be determined. The ligands and / or receptors may be nucleic acids or derivatives thereof (DNA, RNA PNA, LNA, oligonucleotides, plasmids, chromosomes), peptides, proteins (enzyme, protein, oligopeptides, cellular receptor proteins and their complexes, peptide hormones, antibodies and their fragments), Carbohydrates and their derivatives, in particular glycosylated proteins and glycosides, fats, fatty acids and / or lipids.

In vorteilhafter Weise ist es durch die Kombination des kompetitiven Assays mit einem nicht kompetitiven Assay in derselben Messkammer möglich, bei Proben, die mehrere, in ihrer Konzentration stark voneinander abweichende Liganden enthalten, die Konzentrationen der einzelnen Liganden mit nur einem einzigen Versuch zu bestimmen. Die Konzentration des Kompetitors in dem aus der Probe und dem Kompetitor bestehenden Gemisch und die Einwirkungsdauer, während der das Gemisch mit den Rezeptoren in Kontakt steht, werden dabei so auf die für die einzelnen Liganden zu überprüfenden Konzentrationsbereiche abgestimmt, dass nach dem Entfernen der nicht an einen Rezeptor gebundnen Bestandteile des Gemischs an allen Teststellen nur ein Teil der Bindungsstellen Rezeptoren an einen Liganden bindet, wenn dieser in dem zu untersuchenden Konzentrationsbereich in der Probe enthalten ist. Die Konzentration des Kompetitors in dem Gemisch wird vorzugsweise so gewählt, dass sie einem Grenzwert der Konzentration des zweiten Liganden entspricht, aus dem sich eine Aussage, wie z.B. gut/schlecht" oder "krank/gesund", ableiten lässt. Mit dem erfindungsgemäßen Verfahren können in einer Flusszelle oder dergleichen Messkammer nebeneinander Liganden-Konzentrationen in einem Bereich von g/l bis µg/l nachgewiesen werden.Advantageously, by combining the competitive assay with a non-competitive assay in the same measurement chamber, it is possible to determine the concentrations of individual ligands in a single experiment for samples containing multiple ligands that differ greatly in their concentration. The concentration of the competitor in the mixture consisting of the sample and the competitor and the duration of exposure during which the mixture is in contact with the receptors are adjusted to the concentration ranges to be checked for the individual ligands so that after removal of the not a receptor bound components of the mixture at all test sites only a part of the binding sites binds receptors to a ligand, if this is contained in the concentration range to be examined in the sample. The concentration of the competitor in the mixture is preferably selected to correspond to a limit of the concentration of the second ligand from which a statement such as e.g. good / bad "or" sick / healthy. "With the method according to the invention ligand concentrations in a range of g / l to ug / l can be detected side by side in a flow cell or similar measuring chamber.

Bei einer vorteilhaften Ausführungsform der Erfindung wird ein für den ersten Liganden bindungsspezifischer, mit einem ersten Marker markierter Nachweisantikörper mit der ersten Teststelle in Kontakt gebracht, wobei danach nicht an einen immobilisierten Rezeptor gebundene Marker von dem Substrat entfernt werden, und wobei danach das erste Messsignal in Abhängigkeit von der Konzentration des ersten Markers erzeugt wird. Die Konzentration des ersten Liganden kann also mit Hilfe eines Sandwich-Elisa gemessen werden.In an advantageous embodiment of the invention, a detection antibody labeled with a first marker for the first ligand is brought into contact with the first test site, after which markers not bound to an immobilized receptor are removed from the substrate, and then the first measurement signal in Dependent on the concentration of the first marker is generated. The concentration of the first ligand can therefore be measured with the aid of a sandwich Elisa.

Bei einer bevorzugten Ausgestaltung der Erfindung wird der Kompetitor mit einem zweiten Marker markiert, wobei nach dem Entfernen der nicht an einen immobilisierten Rezeptor gebunden Bestandteile des Gemischs von dem Substrat das zweite Messsignal in Abhängigkeit von der Konzentration des an den zweiten Rezeptor gebundenen zweiten Markers erzeugt wird. Dabei kann der zweite Marker mit dem ersten Marker übereinstimmen.In a preferred embodiment of the invention, the competitor is labeled with a second marker, wherein after removing the components of the mixture not bound to an immobilized receptor from the substrate, the second measurement signal is generated as a function of the concentration of the second marker bound to the second receptor , The second marker may coincide with the first marker.

Vorteilhaft ist, wenn der erste Marker und/oder zweite Marker ein Enzym ist (sind), wenn das Enzym während der Erfassung der Messsignale mit mindestens zwei Chemikalien in Kontakt gebracht wird, zwischen denen bei Anwesenheit des Enzyms eine chemische Redoxreaktion auftritt, und wenn das erste Messsignal und/oder das zweite Messsignal durch Messen eines Redoxpotentials erzeugt wird (werden). Dabei kann das Redoxpotential mit Hilfe eines ISFET gemessen werden.It is advantageous if the first marker and / or second marker is (are) an enzyme if the enzyme is at least two during the detection of the measurement signals Chemicals is brought into contact, between which in the presence of the enzyme, a chemical redox reaction occurs, and when the first measurement signal and / or the second measurement signal is generated by measuring a redox potential (be). The redox potential can be measured with the aid of an ISFET.

Bei einer zweckmäßigen Ausgestaltung der Erfindung wird (werde n) der erste Marker und/oder zweite Marker während der Erfassung der Messsignale mit einer Anregungsstrahlung bestrahlt, welche den (die) Marker zur Abgabe einer Lumineszenzstrahlung anregt, wobei das erste Messsignal und/oder das zweite Messsignal durch Messen der Lumineszenzstrahlung des betreffenden Markers erzeugt wird (werden). Dabei kann die Anregungsstrahlung mit Hilfe einer Lichtquelle, beispielsweise einer Leuchtdiode und/oder einer Xenon-Lampe erzeugt werden. Für den ersten und zweiten Marker wird vorzugsweise dieselbe Anregungsstrahlung verwendet. Es ist aber auch denkbar, dass der erste und zweite Marker unterschiedliche Farbstoffe, wie z.B. Cy3 und Cy5 sind, und dass diese Farbstoffe mit unterschiedlichen Wellenlängen angeregt werden.In an expedient embodiment of the invention, the first marker and / or second marker is irradiated during the detection of the measurement signals with an excitation radiation which excites the marker (s) for emitting a luminescence radiation, wherein the first measurement signal and / or the second Measuring signal is generated by measuring the luminescence of the marker in question (be). In this case, the excitation radiation can be generated with the aid of a light source, for example a light-emitting diode and / or a xenon lamp. For the first and second markers, preferably the same excitation radiation is used. However, it is also conceivable that the first and second markers contain different dyes, such as e.g. Cy3 and Cy5, and that these dyes are excited at different wavelengths.

Vorteilhaft ist, wenn die erste Teststelle und/oder zweite Testelle während der Erfassung der Messsignale mit einem Chemilumineszenzsubstrat in Kontakt gebracht wird (werden), in dem in Abhängigkeit von der Bindung des ersten Liganden an den ersten Rezeptor und/oder in Abhängigkeit von der Bindung des Kompetitors an den zweiten Rezeptor zur Abgabe einer Lumineszenzstrahlung angeregt wird, wobei das erste Messsignal und/oder das zweite Messsignal durch Messen der Lumineszenzstrahlung an der betreffenden Teststelle erzeugt wird (werden). Die Lumineszenzstrahlung wird dabei ohne eine Anregungsstrahlung auf chemischem Wege erzeugt.It is advantageous if the first test site and / or second test cell is brought into contact with a chemiluminescent substrate during the detection of the measurement signals, depending on the binding of the first ligand to the first receptor and / or depending on the binding is excited by the competitor to the second receptor for emitting a luminescence, wherein the first measurement signal and / or the second measurement signal is generated by measuring the luminescence at the relevant test site (are). The luminescence radiation is thereby generated without excitation radiation by chemical means.

Die vorstehend genannte Aufgabe wird bezüglich der Vorrichtung dadurch gelöst, dass sie eine Mischeinrichtung aufweist, die zum Vermischen der Probe mit einem für den zweiten Rezeptor bindungsspezifischen Kompetitor mit einer Zuführöffnung für die Probe und einem den Kompetitor enthaltenden Aufnahmeraum verbunden ist, dass die Mischeinrichtung eine Abgabeöffnung für das aus der Probe und dem Kompetitor gebildete Gemisch aufweist, die mit der Einlassöffnung der Messkammer verbunden ist, dass die Mischeinrichtung derart ausgestaltet ist, dass der Kompetitor in einer vorgegebenen Konzentration in dem Gemisch vorliegt, dass der zweite Sensor zum Erfassen eines von der Konzentration des an den zweiten Rezeptor gebundenen Kompetitors abhängigen zweiten Messsignals ausgebildet und mit einer Auswerteeinrichtung verbunden ist, die zum Bestimmen der Konzentration des zweiten Liganden in der Probe aus dem zweiten Messsignal und der Konzentration des Kompetitors in dem Gemisch ausgebildet ist.The above object is achieved with respect to the device in that it comprises a mixing device, which is connected for mixing the sample with a binding agent for the second receptor specific competitor with a feed opening for the sample and a competitor-containing receiving space, that the mixing device is a discharge opening for the mixture formed from the sample and the competitor, which is connected to the inlet opening of the measuring chamber, that the mixing device is designed such that the competitor is present in a predetermined concentration in the mixture, that the second sensor is designed to detect a second measurement signal dependent on the concentration of the competitor bound to the second receptor and connected to an evaluation device for determining the concentration of the second ligand in the sample from the second measurement signal and the concentration of the competitor in the mixture is trained.

Die Probe wird also vor dem Eintritt in die Messkammer mit Hilfe der Mischeinrichtung mit einem für den zweiten Rezeptor bindungsspezifischen Kompetitor vermischt, so dass in der Mischkammer für den zweiten Ligand ein kompetitives Assays und gleichzeitig für den ersten Ligand ein nicht kompetitives Assay durchgeführt werden kann. Wie bereits bei dem Verfahren erläutert wurde, ermöglicht die Kombination dieser Essays bei einer Probe, die mehrere, in ihrer Konzentration stark voneinander abweichende Liganden enthält, die Konzentrationen der einzelnen Liganden mit nur einem einzigen Versuch zu bestimmen.The sample is thus mixed before entering the measuring chamber by means of the mixing device with a binding agent specific for the second receptor competitor, so that in the mixing chamber for the second ligand a competitive assay and simultaneously for the first ligand a non-competitive assay can be performed. As already explained in the method, the combination of these essays in a sample containing a plurality of ligands which differ greatly in their concentration makes it possible to determine the concentrations of the individual ligands in a single experiment.

Vorteilhaft ist, wenn der Kompetitor in gelförmiger, teigiger oder fester Form in dem Aufnahmeraum stabilisiert ist, vorzugsweise derart, dass er an einer Begrenzungswand des Aufnahmeraums anhaftet, und wenn der Aufnahmeraum zum Lösen des Kompetitors in der Probe als Durchflussmischkammer ausgebildet ist, über welche die Zuführöffnung für die Probe mit der Einlassöffnung der Messkammer verbunden ist. Die Vorrichtung ist dadurch besonders gut handhabbar. Die Stabilisierung des Kompetitor umfasst vorzugsweise mindestens ein nicht-reduzierendes Disaccharid und mindestens ein Protein oder Polypeptid der LEA-Klasse. Das nichtreduzierende Disaccharid kann aus einer Gruppe ausgewählt sein, die aus Trehaose (D-Glucopyranosyl-D-glucopyranose), Sucrose (β-D-Fructofüranosyl-α-D-glucopyranosid) sowie Derivaten davon ausgewählt sein kann. Eine derartige Stabilisierung ist in WO 2004/004455 A2 beschrieben. Der in stabilisierter Form vorliegende Kompetitor ist über längere Zeit haltbar.It is advantageous if the competitor is stabilized in a gel-like, doughy or solid form in the receiving space, preferably such that it adheres to a boundary wall of the receiving space, and if the receiving space for releasing the competitor in the sample is designed as a flow mixing chamber, via which the Supply port for the sample is connected to the inlet opening of the measuring chamber. The device is therefore particularly easy to handle. The stabilization of the competitor preferably comprises at least one non-reducing disaccharide and at least one protein or polypeptide of the LEA class. The non-reducing disaccharide may be selected from the group consisting of trehalose (D-glucopyranosyl-D-glucopyranose), sucrose (β-D-fructofuranosyl-α-D-glucopyranoside) and derivatives thereof. Such stabilization is in WO 2004/004455 A2 described. The present in stabilized form competitor is stable for a long time.

Bei einer bevorzugten Ausgestaltung der Erfindung weist die Durchflussmischkammer eine Mischerstruktur auf, die derart ausgestaltet ist, dass das Gemisch beim Durchfluss durch die Durchftussmischkammer vorzugsweise in abwechselnd zueinander entgegengesetzt verlaufende Richtungen abgelenkt wird, und dass die Mischerstruktur zwischen dem in gelförmiger, teigiger oder fester Form vorliegenden Kompetitor und der Einlassöffnung der Messkammer angeordnet ist. Dabei kann der Mischer ein so genannter Möbiusmischer sein, der mit Methoden der Mikrosystemtechnik mit sehr kompakten Abmessungen hergestellt werden kann.In a preferred embodiment of the invention, the flow mixing chamber has a mixer structure which is designed such that the mixture is deflected in alternately opposite directions as it flows through the Durchftussmischkammer, and that the mixer structure between the present in gel, doughy or solid form Competitor and the inlet opening of the measuring chamber is arranged. there The mixer can be a so-called Möbius mixer, which can be produced with methods of microsystems technology with very compact dimensions.

Bei einer zweckmäßigen Ausführungsform der Erfindung sind die Sensoren optische Sensoren, wobei der erste Rezeptor zur Detektion einer in Abhängigkeit von der Bindung des ersten Liganden an den ersten Rezeptor erzeugten ersten Lumineszenzstrahlung vorzugsweise direkt auf dem ersten Sensor und/oder der zweite Rezeptor zur Detektion einer in Abhängigkeit von der Bindung des Kompetitors an den zweiten Rezeptor erzeugten zweiten Lumineszenzstrahlung vorzugsweise direkt auf dem zweiten Sensor angeordnet ist (sind). Die an den Rezeptoren erzeugte Lumineszenzstrahlung kann dann direkt ohne den Umweg über eine Sammellinse in den (die) Sensor(en) eingekoppelt werden.In an expedient embodiment of the invention, the sensors are optical sensors, wherein the first receptor for detecting a first luminescence radiation generated as a function of the binding of the first ligand to the first receptor preferably directly on the first sensor and / or the second receptor for detecting a in Depending on the binding of the competitor to the second receptor generated second luminescent radiation is preferably arranged directly on the second sensor (are). The luminescence radiation generated at the receptors can then be coupled directly into the sensor (s) without the detour via a converging lens.

Vorteilhaft ist, wenn der Kompetitor mit einem Marker markiert ist, der durch Bestrahlung mit einer Anregungsstrahlung zur Abgabe der Lumineszenzstrahlung anregbar ist, wenn die Vorrichtung zum Bestrahlen der zweiten Teststelle mit der Anregungsstrahlung mindestens eine Strahlungsquelle aufweist, und wenn der zweite Sensor für die Lumineszenzstrahlung empfindlich und für die Anregungsstrahlung unempfindlich ist. Dabei kann die Strahlungsquelle auch außerhalb der Messkammer angeordnet sein, wenn diese mindestens einen Wandungsbereich hat, der für die Anregungsstrahlung durchlässig ist.It is advantageous if the competitor is marked with a marker which can be excited by irradiation with an excitation radiation for emitting the luminescence radiation, if the device for irradiating the second test site with the excitation radiation has at least one radiation source, and if the second sensor is sensitive to the luminescence radiation and is insensitive to the excitation radiation. In this case, the radiation source can also be arranged outside the measuring chamber, if it has at least one wall region which is permeable to the excitation radiation.

Die Vorrichtung kann Bestandteil eines Kits nach einem der Ansprüche 12 bis 16 sein, der zusätzlich zu der der Vorrichtung

  • einen mit einem Enzym oder dergleichen Marker markierten, für den ersten Liganden bindungsspezifischen Nachweisantikörper,
  • mindestens zwei Chemikalien, zwischen denen bei einem Kontakt mit dem an dem Nachweisantikörper und/oder dem Kompetitor und/oder dem Nachweisantikörper angeordneten Enzym eine chemische Redoxreaktion auftritt, und/oder
  • ein Chemilumineszenzsubstrat, in dem bei einem Kontakt mit dem an dem Nachweisantikörper und/oder dem Kompetitor angeordneten Enzym eine chemische Reaktion ausgelöst wird, bei der eine Lumineszenzstrahlung freigesetzt wird, und/oder
  • eine Strahlungsquelle, die zur Aussendung der Lumineszenzstrahlung auf die erste Teststelle angeordnet ist,
aufweisen kann. Der Marker, das Chemilumineszenzsubstrat und/oder die Chemikalien können mit einer geeigneten Zuführeinrichtung, wie z.B. einer Mikropumpe oder einer Pipette, der Messkammer zugeführt werden.The device may be part of a kit according to any one of claims 12 to 16, in addition to that of the device
  • a detection antibody labeled with an enzyme or the like, binding-specific for the first ligand,
  • at least two chemicals between which a chemical redox reaction occurs on contact with the enzyme located on the detection antibody and / or the competitor and / or the detection antibody, and / or
  • a chemiluminescent substrate in which, upon contact with the enzyme arranged on the detection antibody and / or the competitor, a chemical reaction is triggered in which a luminescence radiation is released, and / or
  • a radiation source which is arranged to emit the luminescence radiation to the first test site,
can have. The marker, the chemiluminescent substrate and / or the chemicals can be supplied to the measuring chamber with a suitable feed device, such as a micropump or a pipette.

Nachfolgend ist ein Ausführungsbeispiel der Erfindung anhand der Zeichnung näher erläutert. Es zeigen:

Fig. 1
einen Längsschnitt durch eine Vorrichtung zum Bestimmen der Konzentrationen von in einer zu analysierenden Probe enthaltenen Liganden,
Fig. 2
eine schematische Darstellung eines Sandwich ELISA,
Fig. 3
eine schematische Darstellung eines kompetitiven ELISA,
Fig. 4
eine graphische Darstellung von bei zwei Sandwich ELISA ermittelten Messwerten für die Konzentration eines mit Cy5 markierten Liganden, wobei auf der Abszisse die Zeit und auf der Ordinate die Messwertamplitude aufgetragen sind und wobei die Konzentration als Parameter angegeben ist, und
Fig. 5
eine graphische Darstellung von bei drei direkten ELISA ermittelten Messwerten für die Konzentration eines mit Cy5 markierten Liganden, wobei auf der Abszisse die Zeit und auf der Ordinate die Messwertamplitude aufgetragen sind und wobei die Konzentration als Parameter angegeben ist.
An exemplary embodiment of the invention is explained in more detail below with reference to the drawing. Show it:
Fig. 1
a longitudinal section through an apparatus for determining the concentrations of ligands contained in a sample to be analyzed,
Fig. 2
a schematic representation of a sandwich ELISA,
Fig. 3
a schematic representation of a competitive ELISA,
Fig. 4
a graphical representation of measured by two sandwich ELISA measured values for the concentration of a Cy5 labeled ligand, where the abscissa time and plotted on the ordinate the measured value amplitude and wherein the concentration is given as a parameter, and
Fig. 5
a graphical representation of measured values determined by three direct ELISA for the concentration of a ligand labeled with Cy5, wherein the abscissa represents the time and on the ordinate the measured value amplitude and wherein the concentration is given as a parameter.

Eine in Fig. 1 im Ganzen mit bezeichnete Vorrichtung zum Bestimmen der Konzentrationen von mindestens zwei Liganden in einer zu analysierenden Probe weist eine als Flusszelle ausgebildete Messkammer 2 auf, die eine Einlassöffnung 3 und eine Auslassöffnung 4 hat. Eine Wandung der Messkammer 2 ist durch ein Halbleiter-Substrat gebildet, auf dem an einer ersten Teststelle 5 ein erster Rezeptor 6 und an einer seitlich davon beabstandeten zweiten Teststelle 7 ein zweiter Rezeptor 8 immobilisiert sind. Der erste Rezeptor 6 ist für einen ersten, in der zu analysierenden Probe enthaltenen Liganden 9 und der zweite Rezeptor 8 für einen zweiten, in der Probe enthaltenen Liganden bindungsspezifisch. Der zweite Ligand weist eine größere Konzentration in der Probe auf als der erste Ligand 9.An in Fig. 1 As a whole, designated apparatus for determining the concentrations of at least two ligands in a sample to be analyzed comprises a measuring chamber 2 designed as a flow cell, which has an inlet opening 3 and an outlet opening 4. A wall of the measuring chamber 2 is formed by a semiconductor substrate on which a first receptor 6 is immobilized on a first test site 5 and a second receptor 8 is immobilized on a second test site 7 spaced laterally therefrom. The first receptor 6 is for a first, in the analyzed Sample contained ligand 9 and the second receptor 8 for a second, contained in the sample ligand binding specific. The second ligand has a greater concentration in the sample than the first ligand 9.

An der ersten Teststelle 5 ist direkt unter dem ersten Rezeptor 6 ein erster optischer Sensor 10 und an der zweiten Teststelle 7 direkt unter dem zweiten Rezeptor 8 ein zweiter optischer Sensor 11 in das Halbleiter-Substrat integriert. Zusätzlich ist ein dritter optischer Sensor 12 in die Wandung der Messkammer angeordnet, der nicht mit einem Rezeptor bedeckt ist und als Referenzwertgeber dient. Die Sensoren 10, 11, 12 können beispielsweise Photodioden sein.At the first test site 5, a first optical sensor 10 is integrated directly below the first receptor 6 and a second optical sensor 11 is integrated into the semiconductor substrate at the second test site 7 directly below the second receptor 8. In addition, a third optical sensor 12 is arranged in the wall of the measuring chamber, which is not covered with a receptor and serves as a reference value transmitter. The sensors 10, 11, 12 may be photodiodes, for example.

Die Vorrichtung 1 weist ferner eine Mischeinrichtung 1 3 auf, welche die Einlassöffnung 3 der Messkammer 2 mit einer Zuführöffnung 14 verbindet. Die Mischeinrichtung 13 dient dazu, die Probe mit einem für den zweiten Rezeptor 8 bindungsspezifischen Kompetitor 15 zu vermischen, der mit einem Enzym 16, wie z.B. HRP (Meerrettich-Peroxidase), markiert ist. Dazu wird die Probe beispielsweise mit Hilfe einer Pipette oder einer Pumpe durch die Zuführöffnung 14 hindurch in einen Aufnahmeraum 16 der Mischeinrichtung 13 eingeleitet. In dem Aufnahmeraum 17 ist der markierte Kompetitor 15 in stabilisierter Form derart relativ zu der Zuführöffnung 14 angeordnet, dass die Probe während und/oder nach dem Einleiten in den Aufnahmeraum 17 mit dem Kompetitor 15 bzw. dem damit verbundenen Enzym 16 in Kontakt gerät und sich mit den Kompetitor-Enzym-Komplexen vermischt. In Strömungsrichtung hinter dem Aufnahmeraum 17 weist die Mischeinrichtung 1 3 eine als Möbiusmischer ausgestaltete Mischerstruktur 18 auf, die mit ihrem einen Ende mit dem Aufnahmeraum 17 und mit ihrem anderen, eine Abgabeöffnung für das aus der Probe und den Kompetitor-Enzym-Komplexen gebildete Gemisch aufweisenden Ende über einen Kanal 19 mit der Einlassöffnung 3 der Messkammer 2 verbunden ist.The device 1 further comprises a mixing device 1 3, which connects the inlet opening 3 of the measuring chamber 2 with a feed opening 14. The mixing means 13 serves to mix the sample with a second receptor-specific competitor 15 which is bound to an enzyme 16, such as an enzyme. HRP (horseradish peroxidase) is marked. For this purpose, the sample is introduced, for example with the aid of a pipette or a pump through the feed opening 14 into a receiving space 16 of the mixing device 13. In the receiving space 17, the marked competitor 15 is arranged in a stabilized form relative to the feed opening 14 such that the sample comes into contact with the competitor 15 or the associated enzyme 16 during and / or after being introduced into the receiving space 17 mixed with the competitor-enzyme complexes. In the flow direction behind the receiving space 17, the mixing device 1 3 has a mixer structure 18 configured as a Möbius mixer, which with its one end having the receiving space 17 and with their other, a discharge opening for the mixture formed from the sample and the competitor-enzyme complexes End is connected via a channel 19 with the inlet opening 3 of the measuring chamber 2.

Die Mischeinrichtung 13 ist derart ausgestaltet, dass der Kompetitor 15 in der Mischkammer 2 in einer vorgegebenen, einem zu messenden Grenzwert entsprechenden Konzentration in dem Gemisch vorliegt. Dies wird dadurch erreicht, dass die Messkammer 2 und die Mischeinrichtung 13 ein vorbestimmtes Volumen aufweisen, und dass die Menge des in dem Aufnahmeraum 17 stabilisierten Kompetitors 15 so gewählt ist, dass sich die gewünschte Konzentration einstellt, wenn der Kompetitor 15 mit der Probe zu einem Gemisch vermischt wird, welches das vorbestimmte Volumen aufweist.The mixing device 13 is configured in such a way that the competitor 15 is present in the mixing chamber 2 at a predetermined concentration in the mixture corresponding to a limit value to be measured. This is achieved in that the measuring chamber 2 and the mixing device 13 have a predetermined volume, and that the amount of competitor 15 stabilized in the receiving space 17 is selected such that the desired concentration is established, when the competitor 15 is mixed with the sample to a mixture having the predetermined volume.

Nachdem die Messkammer 2 mit dem aus der Probe und den Kompetitor-Enzym-Komplexen gebildeten Gemisch befüllt wurde, wird eine vorgegebene erste Zeitdauer abgewartet, um den in dem Gemisch enthaltenen Liganden und dem Kompetitor 15 die Möglichkeit zu geben, jeweils an die für sie bindungsspezifischen Rezeptoren 6, 8 zu binden. Die erste Zeitdauer wird so gewählt, dass jeweils nur ein Teil der an den einzelnen Teststellen 5, 7 vorhanden freien Bindungsstellen der Rezeptoren 6, 8 an einen Liganden bindet. In Fig. 2 ist das Binden eines Moleküls des ersten Liganden 9 an den ersten Rezeptor 6 und in Fig. 3 das Binden eines Kompetitor-Enzym-Komplexes an den zweiten Rezeptor 8 schematisch dargestellt.After the measuring chamber 2 has been filled with the mixture formed from the sample and the competitor-enzyme complexes, a predetermined first period of time is waited to give the ligands contained in the mixture and the competitor 15 the possibility to bind to each of them To bind receptors 6, 8. The first time period is selected such that in each case only part of the free binding sites of the receptors 6, 8 present at the individual test sites 5, 7 bind to a ligand. In Fig. 2 is the binding of a molecule of the first ligand 9 to the first receptor 6 and in FIG Fig. 3 the binding of a competitor-enzyme complex to the second receptor 8 is shown schematically.

Nachdem die vorgegebene erste Zeitdauer abgelaufen ist, wird über die Zuführöffnung 14, die Einlassöffnung 3 und die Auslassöffnung 4 eine Spülflüssigkeit durch die Messkammer 2 hindurchgeleitet, welche die nicht an einen Rezeptor 6, 8 gebundenen Bestandteile des gebildeten Gemischs aus der Messkammer 2 entfernt.After the predetermined first period of time has elapsed, a rinsing liquid is passed through the measuring chamber 2 via the feed opening 14, the inlet opening 3 and the outlet opening 4, which removes the components of the mixture formed from the measuring chamber 2 which are not bound to a receptor 6, 8.

Danach wird eine Lösung in die Messkammer 2 eingebracht, die einen Nachweisantikörper 20 enthält, der für den ersten Liganden 9 bindungsspezifisch ist und mit einem Enzym 21, wie z.B. HRP, markiert ist. Nachdem der Nachweisantikörper 20 in die Messkammer 2 eingebracht wurde, wird eine vorgegebene zweite Zeitdauer abgewartet, die so gewählt ist, dass nahezu alle an den ersten Rezeptor 6 gebundenen Moleküle des ersten Liganden 9 jeweils an ein Molekül des Nachweisantikörpers 20 binden und dadurch indirekt mit dem Enzym 21 markiert werden.Thereafter, a solution is introduced into the measuring chamber 2 which contains a detection antibody 20 which is specific for the first ligand 9 and which is bound with an enzyme 21, e.g. HRP, marked. After the detection antibody 20 has been introduced into the measuring chamber 2, a predetermined second time period is waited, which is selected so that almost all bound to the first receptor 6 molecules of the first ligand 9 each bind to a molecule of the detection antibody 20 and thereby indirectly with the Enzyme 21 can be marked.

Nachdem die vorgegebene zweite Zeitdauer abgelaufen ist, wird erneut die Spülflüssigkeit durch die Messkammer 2 hindurchgeleitet, um nicht an einen ersten Liganden gebundene Nachweisantikörper 20 aus der Messkammer 2 zu entfernen.After the predetermined second period of time has elapsed, the rinsing liquid is again passed through the measuring chamber 2, in order not to remove detection antibodies 20 bound to a first ligand from the measuring chamber 2.

Danach wird über die Zuführöffnung 14 und die Einlassöffnung 3 ein Chemi-Lumineszenzsubstrat in die Messkammer 2 eingeleitet, das Wasserstoffperoxid und einen Chemiluminophor, wie z.B. Luminol, enthält. Wenn die Enzyme 16, 21 mit dem Wasserstoffperoxid in Kontakt geraten, werden freie Sauerstoffradikale von dem Wasserstoffperoxid abgespalten, wodurch der Chemiluminophor unter Abgabe von Lumineszenzstrahlung chemisch zersetzt wird. Die Lumineszenzstrahlung wird also einerseits in Abhängigkeit von der Bindung des ersten Liganden an den ersten Rezeptor 6 und andererseits in Abhängigkeit von der Bindung des Kompetitors 15 an den zweiten Rezeptor 8 in der Messkammer 2 erzeugt.Thereafter, via the feed opening 14 and the inlet opening 3, a chemiluminescent substrate is introduced into the measuring chamber 2 containing hydrogen peroxide and a chemiluminophore such as luminol. When the enzymes 16, 21 with the Hydrogen peroxide come into contact, free oxygen radicals are cleaved from the hydrogen peroxide, whereby the chemiluminophore is chemically decomposed under the emission of luminescence radiation. The luminescence radiation is thus generated on the one hand as a function of the binding of the first ligand to the first receptor 6 and on the other hand as a function of the binding of the competitor 15 to the second receptor 8 in the measuring chamber 2.

Die optischen Sensoren 10, 11 sind für die Lumineszenzstrahlung empfindlich und derart relativ zu den Rezeptoren 6, 8 angeordnet, dass sie jeweils nur die an der ihnen zugeordneten Teststelle 5, 7 erzeugte Lumineszenzstrahlung detektieren, nicht jedoch die Lumineszenzstrahlung, die an der jeweils anderen Teststelle 7, 5 erzeugt wird.The optical sensors 10, 11 are sensitive to the luminescence radiation and arranged relative to the receptors 6, 8 such that they respectively detect only the luminescence radiation generated at their associated test site 5, 7, but not the luminescence radiation at the respective other test site 7, 5 is generated.

Mit den Sensoren 10, 11 ist eine in der Zeichnung nicht näher dargestellte Auswerteeinrichtung verbunden, die in Abhängigkeit von dem Messsignal des ersten Sensors 10 die Konzentrationen des ersten Liganden 6 in der Probe und in Abhängigkeit von dem Messsignal des zweiten Sensors 11 und der bekannten Konzentration des Kompetitors 15 in dem Gemisch mit Hilfe des Massenwirkungsgesetzes die Konzentration des zweiten Liganden in der Probe bestimmt. Die Auswerteeinrichtung kann als elektrische Schaltung in das Substrat bzw. die Wandung der Messkammer 2 integriert sein.With the sensors 10, 11 an evaluation device not shown in the drawing is connected, the concentrations of the first ligand 6 in the sample and in dependence on the measurement signal of the second sensor 11 and the known concentration depending on the measurement signal of the first sensor of the competitor 15 in the mixture determined by means of the law of mass action, the concentration of the second ligand in the sample. The evaluation device can be integrated as an electrical circuit in the substrate or the wall of the measuring chamber 2.

In Fig. 4 und 5 sind mit einem direkten ELISA und einem Sandwich ELISA ermittelte Messwerte als Funktion der Zeit graphisch dargestellt. Bei dem ELISA wird in Abhängigkeit von der Bindung des ersten Liganden an den einen Rezeptor und in Abhängigkeit von der Bindung des zweiten Liganden an einen anderen Rezeptor jeweils eine Lumineszenzstrahlung erzeugt. Die Liganden werden dazu mit Hilfe eines Markers, wie z.B. dem Farbstoff Cy5, markiert und mit einer Anregungsstrahlung bestrahlt, bei welcher der Marker zur Abgabe von Lumineszenzstrahlung angeregt wird. Deutlich ist erkennbar, dass der Anstieg der Messwerte mit abnehmender Konzentration der Liganden in der Probe flacher wird. Durch das erfindungsgemäße Verfahren wird es möglich, die Konzentration zweier in einer Probe in stark unterschiedlicher Konzentration enthaltener Liganden gleichzeitig in der Messkammer 2 zu messen. Dies wird dadurch erreicht, dass die Konzentration des die hohe Konzentration aufweisenden Liganden mit Hilfe eines kompetitiven Assays und die Konzentration des die geringe Konzentration aufweisenden Liganden 9 mit Hilfe eines nicht kompetitiven Assays gemessen werden, so dass bei der Messsignalgewinnung keiner der Sensoren 10, 11 in die Begrenzung gerät.In Fig. 4 and 5 For example, readings taken with a direct ELISA and a sandwich ELISA are plotted as a function of time. In the case of the ELISA, depending on the binding of the first ligand to one receptor and depending on the binding of the second ligand to another receptor, in each case a luminescence radiation is generated. The ligands are for this purpose marked with the aid of a marker, such as the dye Cy5, and irradiated with an excitation radiation, in which the marker is excited to emit luminescence radiation. It can be clearly seen that the increase in the measured values becomes flatter with decreasing concentration of the ligands in the sample. By means of the method according to the invention, it is possible to measure the concentration of two ligands contained in a sample in greatly differing concentrations simultaneously in the measuring chamber 2. This is achieved by increasing the concentration of the high concentration ligand using a competitive assay and the concentration of the low-concentration ligand 9 are measured by means of a non-competitive assay, so that none of the sensors 10, 11 gets into the limit during measurement signal acquisition.

Claims (16)

  1. Method for determining the concentrations of at least two ligands supposed to be present in a sample to be analysed, wherein a first ligand (9) binds specifically to a first receptor (6) and a second ligand binds specifically to a second receptor (8), wherein the first receptor (6) is immobilized to at least one first test site (5) and the second receptor (8) is immobilized to at least one second test site (7) on a substrate, wherein a competitor (15) that binds specifically to the second receptor (8) is mixed with the sample, wherein the mixture is contacted with the substrate in such a way that the first ligand (9) can bind to the first receptor (6) and the second ligand and/or the competitor (15) can bind to the second receptor (8), wherein components of the mixture, which are not bound to an immobilized receptor (6, 8), are subsequently removed from the substrate, wherein a first measurement signal and a second measurement signal which is a function of the concentration of the competitor (15) bound to the second receptor (8) are generated, characterized in that the competitor (15) that binds specifically to the second receptor (8) is mixed with the sample in such a way that the competitor (15) is present at a predefined concentration in the mixture thus obtained, that the first measurement signal is generated in such a way that it is a function of the concentration of the first ligand (9) bound to the first receptor (6), and that the concentration of the first ligand (9) in the sample is determined with the aid of the first measurement signal and the concentration of the second ligand in the sample is determined with the aid of the second measurement signal and the known concentration of the competitor (15) in the mixture.
  2. Method according to Claim 1, characterized in that a detection antibody (20) that binds specifically to the first ligand (9) and has been labelled with a first marker is contacted with the first test site (5), that markers not bound to an immobilized receptor (56, 8) are subsequently removed from the substrate, and that the first measurement signal is then generated as a function of the concentration of the first marker.
  3. Method according to Claim 1 or 2, characterized in that the competitor (15) is labelled with a second marker, and that, after the components of the mixture that are not bound to an immobilized receptor (6, 8) have been removed from the substrate, the second measurement signal is generated as a function of the concentration of the second marker bound to the second receptor (8).
  4. Method according to any of Claims 1 to 3, characterized in that the first marker and/or the second marker is (are) an enzyme (16, 21), that, while the measurement signals are recorded, the enzyme (16, 21) is contacted with at least two chemicals which undergo a chemical redox reaction with one another in the presence of the enzyme, and that the first measurement signal and/or the second measurement signal are (is) generated by measuring a redox potential.
  5. Method according to any of Claims 1 to 4, characterized in that, while the measurement signals are recorded, the first marker and/or the second marker are (is) irradiated with an excitation radiation which causes the marker(s) to emit a luminescent radiation, and that the first measurement signal and/or the second measurement signal are (is) generated by measuring the luminescent radiation of the relevant marker.
  6. Method according to any of Claims 1 to 5, characterized in that, while the measurement signals are recorded, the first test site (5) and/or the second test site (7) are (is) contacted with a chemiluminescent substrate in which excitation to cause the emission of a luminescent radiation takes place as a function of the first ligand (9) binding to the first receptor (6) and/or as a function of the competitor (15) binding to the second receptor (8), and that the first measurement signal and/or the second measurement signal are (is) generated by measuring the luminescent radiation at the relevant test site (5, 7).
  7. Apparatus (1) for determining the concentrations of at least two ligands in a sample to be analyzed, having a measurement chamber (2) which has at least one inlet orifice (3) and one outlet orifice (4) and has a substrate on which a first receptor (6) is immobilized at a first test site (5) and a second receptor (8) is immobilized at a second test site (7), wherein the first receptor (6) binds specifically to a first ligand (9) and the second receptor (8) binds specifically to a second ligand, wherein a first sensor (10) for recording a first measurement signal as a function of the concentration of the ligand bound to the first receptor (6) has been assigned to the first test site (5) and a second sensor (11) has been assigned to the second test site (7), characterized in that the apparatus (1) has a mixing device (13) which is connected to a supply orifice (14) for the sample and to a reception space (17) containing a competitor (15) that binds specifically to the second receptor (8), in order to mix the sample with the competitor (15), that the mixing device (13) has a discharge orifice for the mixture of the sample and the competitor (15), which discharge orifice is connected to the inlet orifice (3) of the measurement chamber (2), that the mixing device (13) is designed for the competitor (15) to be present at a predefined concentration in the mixture, that the second sensor (11) is designed for recording a second measurement signal as a function of the concentration of the competitor (15) bound to the second receptor (8) and is connected to an evaluation device designed for determining the concentration of the second ligand in the sample from the second measurement signal and the concentration of the competitor (15) in the mixture.
  8. Apparatus (1) according to Claim 7, characterized in that the competitor (15) is stabilized in a gel-like, doughy or solid form in the reception space (17), preferably so as to adhere to a boundary wall of the reception space (17), and that the reception space (17) is designed as a mixing flow chamber for dissolving the competitor (15) in the sample, via which chamber the supply orifice (14) for the sample is connected to the inlet orifice (3) of the measurement chamber (2).
  9. Apparatus (1) according to Claim 7 or 8, characterized in that the mixing flow chamber has a mixing structure (18) which is equipped in such a way that the mixture flowing through the mixing flow chamber is deflected preferably in alternately opposite directions, and that the mixing structure (18) is arranged between the gel-like, doughy or solid competitor (15) and the inlet orifice (3) of the measurement chamber (2).
  10. Apparatus (1) according to any of Claims 7 to 9, characterized in that the sensors (10, 11) are optical sensors, and that the first receptor (6) for detecting a first luminescent radiation generated as a function of the first ligand (9) binding to the first receptor (6) is arranged preferably directly on the first sensor (10) and/or the second receptor (8) for detecting a second luminescent radiation generated as a function of the competitor (15) binding to the second receptor (8) is arranged preferably directly on the second sensor (11).
  11. Apparatus (1) according to any of Claims 7 to 10, characterized in that the competitor (15) is labelled with a marker which can be excited by irradiating with an excitation radiation in order to emit the luminescent radiation, that the apparatus (1) has at least one radiation source for irradiating the second test site (7) with the excitation radiation, and that the second sensor (11) is sensitive to the luminescent radiation and insensitive to the excitation radiation.
  12. Kit for carrying out the method according to any of Claims 1 to 6, comprising
    - an apparatus (1) according to any of Claims 7 to 11, in which the first sensor (10) is a sensor for measuring a redox potential,
    - a detection antibody (20) binding specifically to the first ligand (9) and labelled with an enzyme (21), and
    - at least two chemicals which undergo a chemical redox reaction with one another when contacting the enzyme.
  13. Kit for carrying out the method according to any of Claims 1 to 6, particularly according to Claim 12, comprising
    - an apparatus (1) according to any of Claims 7 to 11,
    - a detection antibody (20) binding specifically to the first ligand (9) and labelled with an enzyme (21), and
    - a chemiluminescent substrate in which, upon contact with the enzyme (21), a chemical reaction is triggered, releasing a luminescent radiation to which the first sensor (10) is sensitive.
  14. Kit for carrying out the method according to any of Claims 1 to 6, particularly according to Claim 12 or 13, comprising
    - a detection antibody (20) binding specifically to the first ligand (9) and labelled with a marker which can be excited by irradiating with an excitation radiation in order to emit a luminescent radiation,
    - an apparatus (1) according to any of Claims 7 to 11, in which the first sensor (10) is sensitive to the luminescent radiation and insensitive to the excitation radiation, and
    - a radiation source which is arranged for emitting the luminescent radiation to the first test site (5).
  15. Kit for carrying out the method according to any of Claims 1 to 6, particularly according to any of Claims 12 to 14, comprising
    - an apparatus (1) according to any of Claims 7 to 11, in which the competitor (15) is labelled with an enzyme (16) and in which the second sensor (11) is designed for measuring an electric redox potential, and
    - at least two chemicals which undergo a chemical redox reaction with one another when contacting the enzyme (16).
  16. Kit for carrying out the method according to any of Claims 1 to 6, particularly according to any of Claims 12 to 15, comprising
    - an apparatus (1) according to any of Claims 7 to 11, in which the competitor (15) is labelled with an enzyme (16), and
    - a chemiluminescent substrate in which, upon contact with the enzyme (16), a chemical reaction is triggered, releasing a luminescent radiation to which the second sensor (11) is sensitive.
EP05007284A 2005-04-04 2005-04-04 Method and apparatus for determining the concentrations of at least 2 ligands. Not-in-force EP1715341B1 (en)

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DE502005002994T DE502005002994D1 (en) 2005-04-04 2005-04-04 Method and apparatus for determining the concentrations of at least two ligands
EP05007284A EP1715341B1 (en) 2005-04-04 2005-04-04 Method and apparatus for determining the concentrations of at least 2 ligands.
US11/397,347 US7598044B2 (en) 2005-04-04 2006-04-04 Procedure and device for determining the concentrations of at least two ligands
US12/558,750 US20100003702A1 (en) 2005-04-04 2009-09-14 Procedure And Device For Determining The Concentrations Of At Least Two Ligands

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Publication number Priority date Publication date Assignee Title
CN104056567A (en) * 2014-06-30 2014-09-24 张帅 Oscillation evenly-shaking device for chemical reagents
CN104056567B (en) * 2014-06-30 2016-08-24 张帅 A kind of chemical reagent vibration oscillating uniform device

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EP1715341A1 (en) 2006-10-25

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